PROJECT SUMMARY/ABSTRACT The Vanderbilt Mouse Metabolic Phenotyping Center (VMMPC) is developing a range of new analytical services based on the use of stable isotope labeling to measure metabolic pathway fluxes. A single quadrupole GC-MS is currently used to measure 13C and 2H enrichments in plasma glucose. However, there is a growing demand for analysis of stable isotope enrichment in organic/amino acids, glycerol, bile acids, ketone bodies, lipids, and cholesterol. Some of this demand has been created by the closure of the MMPC at Case Western Reserve University, which previously collaborated closely with the VMMPC to provide these measurements. This proposal seeks to fill an important void in the VMMPC research infrastructure through the acquisition of an Agilent gas chromatograph-triple quadrupole tandem mass spectrometer (GC-QqQ MS). There are currently no open-access GC-QqQ instruments on the Vanderbilt campus. The requested instrument will enable the VMMPC to expand its service offerings without the additional downtime required to switch its existing GC-MS system, which is already utilized heavily, between different assay configurations. Triple quadrupole GC-QqQ instruments have at least two distinct advantages over single quadrupole instruments when applied to measure stable isotope enrichment. First, reduced interference leads to increased sensitivity and selectivity when detecting dilute components within complex biological samples. As a result, isotope ratios determined by GC- QqQ exhibit enhanced signal-to-noise ratio and dynamic range, which enables precise quantification of low isotope enrichments that are typically obtained from in vivo tracer studies of animals and human subjects. Second, additional product ions produced by collision-induced dissociation provide increased positional labeling information that improves the accuracy of flux determination by stable isotope tracer methods. These capabilities will not only streamline VMMPC workflows but will enable novel technologies to be developed that leverage tandem MS technology to increase the precision of flux measurements, expand pathway coverage, and decrease tracer costs. The instrument will be operated by a dedicated technician who will be trained to prepare and analyze samples. Users will request services using established VMMPC procedures for ordering, billing, and data reporting. The instrument will be placed into an existing VMMPC Core laboratory at Vanderbilt that is already well versed in GC-MS management and applications, so the impact on a wide body of NIH- funded investigators who use animal models to study metabolism will be immediate and substantial.